Muscle Fibrosis Research Articles

Rotator cuff muscle degeneration in a mouse model of glenohumeral osteoarthritis induced by monoiodoacetic acid.

Osteoarthritis (OA) is a disease of joint degeneration and impaired function. Muscle atrophy, fatty infiltration (FI), and fibrosis are degenerative features of muscle injury and predict poor outcomes in some degenerative and exercise-related injuries. Patients with glenohumeral joint OA are usually accompanied by rotator cuff muscle degeneration, even though the rotator cuff is intact. However, the mechanism and correlation between OA and degeneration of muscles around joints are still unknown. Forty-five 12-month-old C57BL/6J mice received a single injection of monoiodoacetic acid(MIA)into the right glenohumeral joint. The sham group was injected with saline on the same day in the right glenohumeral joint. 3 and 6 weeks after the operation, gait analysis was conducted to evaluate the function of the forelimb. Then, the shoulder joint and supraspinatus muscle (SS) were collected for histological staining, RT-qPCR, and biomechanics test. Correlations between fat area fraction in muscle, percentage wet muscle weight change or OARSI score and gait analysis,/muscle mechanics tests were assessed using Pearson's correlation coefficient or Spearman's correlation coefficient. Compared with the sham group, the MIA group developed significant glenohumeral joint OA, and the SS developed significant FI and muscle atrophy. Shoulder function correlated with glenohumeral joint OA/rotator cuff muscle severity, weight loss, and FI. In mice, glenohumeral joint OA can lead to rotator cuff degeneration and inferior limb function. The small animal model could be a powerful tool to further study the potential mechanisms between glenohumeral OA and rotator cuff muscle degeneration. Basic Science Study; In-Vivo Animal Model.

PTEN-AKT pathway attenuates apoptosis and adverse remodeling in ponatinib-induced skeletal muscle toxicity following BMP-7 treatment.

Tyrosine kinase inhibitors (TKIs) including ponatinib are commonly used to treat cancer patients. Unfortunately, TKIs induce cardiac as well as skeletal muscle dysfunction as a side effect. Therefore, detailed mechanistic studies are required to understand its pathogenesis and to develop a therapeutic treatment. The current study was undertaken to examine whether ponatinib induces apoptosis and apoptotic mechanisms both in vitro and in vivo models and furthermore to test the potential of bone morphogenetic protein 7 (BMP-7) as a possible treatment option for its attenuation. Sol8 cells, a mouse myogenic cell line was exposed to ponatinib to generate an apoptotic cell culture model and were subsequently treated with BMP-7 to understand its protective effects. For the in vivo model, C57BL/6J mice were administered with ponatinib to understand apoptosis, cell signaling apoptotic mechanisms, and adverse muscle remodeling and its attenuation with BMP-7. TUNEL staining, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) methods were used. Our data show significantly (p < 0.05) increased TUNEL staining, caspase-3, BAX/Bcl2 ratio in the in vitro model. Furthermore, our in vivo muscle data show ponatinib-induced muscle myopathy, and loss in muscle function. The observed muscle myopathy was associated with increased apoptosis, caspase-3 staining, and BAX/Bcl-2 ratio as confirmed with IHC and RT-PCR. Furthermore, our data show a significant (p < 0.05) increase in the involvement of cell signaling apoptotic regulator protein PTEN and a decrease in cell survival protein AKT. These results suggest that increased apoptosis following ponatinib treatment showed an increase in skeletal muscle remodeling, sarcopenia, and fibrosis. Furthermore, BMP-7 treatment significantly (p < 0.05) attenuated ponatinib-induced apoptosis, BAX/Bcl2 ratio, decreased PTEN, and increased cell survival protein AKT, decreased adverse muscle remodeling, and improved muscle function. Overall, we provide evidence that ponatinib-induces apoptosis leading to sarcopenia and muscle myopathy with decreased function which was attenuated by BMP-7.

Cellular interplay in skeletal muscle regeneration and wasting: insights from animal models.

Skeletal muscle wasting, whether related to physiological ageing, muscle disuse or to an underlying chronic disease, is a key determinant to quality of life and mortality. However, cellular basis responsible for increased catabolism in myocytes often remains unclear. Although myocytes represent the vast majority of skeletal muscle cellular population, they are surrounded by numerous cells with various functions. Animal models, mostly rodents, can help to decipher the mechanisms behind this highly dynamic process, by allowing access to every muscle as well as time-course studies. Satellite cells (SCs) play a crucial role in muscle regeneration, within a niche also composed of fibroblasts and vascular and immune cells. Their proliferation and differentiation is altered in several models of muscle wasting such as cancer, chronic kidney disease or chronic obstructive pulmonary disease (COPD). Fibro-adipogenic progenitor cells are also responsible for functional muscle growth and repair and are associated in disease to muscle fibrosis such as in chronic kidney disease. Other cells have recently proven to have direct myogenic potential, such as pericytes. Outside their role in angiogenesis, endothelial cells and pericytes also participate to healthy muscle homoeostasis by promoting SC pool maintenance (so-called myogenesis-angiogenesis coupling). Their role in chronic diseases muscle wasting has been less studied. Immune cells are pivotal for muscle repair after injury: Macrophages undergo a transition from the M1 to the M2 state along with the transition between the inflammatory and resolutive phase of muscle repair. T regulatory lymphocytes promote and regulate this transition and are also able to activate SC proliferation and differentiation. Neural cells such as terminal Schwann cells, motor neurons and kranocytes are notably implicated in age-related sarcopenia. Last, newly identified cells in skeletal muscle, such as telocytes or interstitial tenocytes could play a role in tissular homoeostasis. We also put a special focus on cellular alterations occurring in COPD, a chronic and highly prevalent respiratory disease mainly linked to tobacco smoke exposure, where muscle wasting is strongly associated with increased mortality, and discuss the pros and cons of animal models versus human studies in this context. Finally, we discuss resident cells metabolism and present future promising leads for research, including the use of muscle organoids.

Guanidinium-Rich Lipopeptide-Based Nanoparticle Enables Efficient Gene Editing in Skeletal Muscles

Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.

Resistance andendurance exercisetraining improves muscle mass and the inflammatory/fibrotic transcriptome in a rhabdomyosarcoma model.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that most often develops in children. Chemoradiation therapy is a standard treatment modality; however, the detrimental long-term skeletal muscle consequences of this therapy in juvenile cancer survivors include muscle atrophy and fibrosis resulting in decreased physical performance. Using a novel model of murine resistanceand endurance exercisetraining, we investigate its role in preventing the long-term effects of juvenile RMS plus therapy. Four-week-old male (n=10) and female (n=10) C57Bl/6J mice were injected with M3-9-M RMS cell into the left gastrocnemius with the right limb serving as an internal control (CON). Mice received a systemic vincristine injection and then five doses of 4.8Gy of gamma radiation localized to the left hindlimb (RMS+Tx). Mice were then randomly divided into either sedentary (SED) or resistanceand endurance exercisetraining (RET) groups. Changes in exercise performance, body composition, myocellular adaptations and the inflammatory/fibrotic transcriptome were assessed. RET improved endurance performance (P<0.0001) and body composition (P=0.0004) compared to SED. RMS+Tx resulted in significantly lower muscle weight (P=0.015) and significantly smaller myofibre cross-sectional area (CSA) (P=0.014). Conversely, RET resulted in significantly higher muscle weight (P=0.030) and significantly larger Type IIA (P=0.014) and IIB (P=0.015) fibre CSA. RMS+Tx resulted in significantly more muscle fibrosis (P=0.028), which was not prevented by RET. RMS+Tx resulted in significantly fewer mononuclear cells (P<0.05) and muscle satellite (stem) cells (MuSCs) (P<0.05) and significantly more immune cells (P<0.05) than CON. RET resulted in significantly more fibro-adipogenic progenitors (P<0.05), a trend for more MuSCs (P=0.076) than SED and significantly more endothelial cells specifically in the RMS+Tx limb. Transcriptomic changes revealed significantly higher expression of inflammatory and fibrotic genes in RMS+Tx, which was prevented by RET. In the RMS+Tx model, RET also significantly altered expression of genes involved in extracellular matrix turnover. Our study suggests that RET preserves muscle mass and performance in a model of juvenile RMS survivorship while partially restoring cellular dynamics and the inflammatory and fibrotic transcriptome.

Crouzon syndrome complicated with binocular strabismus and extraocular muscle fibrosis: a case report

BackgroundCrouzon syndrome, a rare genetic disorder characterized by premature closure of coronal sutures, results in skull and facial deformities along with abnormal brain and ocular development.Case presentationHere, we report a case of a 27-year-old ethnic han male patient who presented with complex binocular strabismus secondary to Crouzon syndrome. At the time of surgery, extraocular muscles were found to be fibrotic and results of the pathological examination revealed degeneration of muscle fibers, which were replaced by adipose tissue. The entire exome sequencing DNA testing indicated that the patient and his father possessed the fibroblast growth factor receptor 2 (FGFR2) gene c.G812T:p.G271V heterozygous mutation. Binocular strabismus corrective surgery was performed in this patient with a satisfactory outcome.ConclusionsThis case demonstrates that Crouzon syndrome patients can show an FGFR2 gene c.G812T:p.G271V mutation and display clinical symptoms such as extraocular muscle fibrosis, exotropia, exophthalmos, and a pointed head deformity.

DNase I and Sivelestat Ameliorate Experimental Hindlimb Ischemia-Reperfusion Injury by Eliminating Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) play an important role in ischemia-reperfusion injury (IRI) of the hindlimb. The aim of this study was to investigate the effect of recombinant DNase I and sivelestat in eliminating NETs and their effects on IRI limbs. An air pump was used to apply a pressure of 300 mmHg to the root of the right hindlimb of the rat for 2 h and then deflated to replicate the IRI model. The formation of NETs was determined by the detection of myeloperoxidase (MPO), neutrophil elastase (NE), and histone H3 in the skeletal muscles of the hindlimbs. Animals were administered 2.5 mg/kg bw/d DNase I, 15 or 60 mg/kg bw/d sivelestat by injection into the tail vein or intramuscularly into the ischemic area for 7d. Elimination of NETs, hindlimb perfusion, muscle fibrosis, angiogenesis and motor function were assessed. DNase I reduced NETs, attenuated muscle fibrosis, promoted angiogenesis in IRI area and improved limb motor function. Local administration of DNase I improved hindlimb perfusion more than intravenous administration. Sivelestat at a dose of 15 mg/kg bw/d increased perfusion, counteracted skeletal muscle fibrosis, promoted angiogenesis and enhanced motor function. However, sivelestat at a dosage of 60 mg/kg bw/d had an adverse effect on tissue repair, especially when injected locally. Both DNase I and moderate doses of sivelestat can eliminate IRI-derived NETs. They improve hindlimb function by improving perfusion and angiogenesis, preventing muscle fibrosis. Appropriate administration mode and dosage is the key to prevent IRI by elimination of NETs. DNase I is more valid when administered topically and sivelestat is more effective when administered intravenously. These results will provide a better strategy for the treatment of IRI in clinical.

BMP-7 Attenuates Sarcopenia and Adverse Muscle Remodeling in Diabetic Mice via Alleviation of Lipids, Inflammation, HMGB1, and Pyroptosis.

Diabetic myopathy involves hyperglycemia, oxidative stress, and inflammation. However, the role of hypercholesterolemia-induced inflammation-mediated pathological mechanisms leading to fibrosis, sarcopenia, deterioration of muscle, and muscle dysfunction in diabetes is not well understood. In this study, we investigated the novel role of bone morphogenetic protein-7 (BMP-7) in ameliorating metabolic alterations, inflammation, pyroptosis, TGF-β/SMAD cell signaling mechanisms, and progression of diabetic myopathy. C57BL/6J mice were treated with saline, streptozotocin (STZ), or STZ+BMP-7. Diabetes was confirmed by increased fasting glucose levels and a glucose tolerance test. Gastrocnemius muscle and blood samples were collected for lipid and tissue analysis using various methods. A significant increase in hyperglycemia resulted in an increase in lipid accumulation, monocyte infiltration, and inflammation, as well as an increase in pyroptotic markers and signaling markers in diabetic muscle myocytes. A structural analysis showed significant muscle loss, and increased muscle deterioration and fibrosis leading to muscle dysfunction. BMP-7 attenuated pathological processes that resulted in significantly improved muscle function. We report, for the first time, that increased hyperlipidemia aggravates inflammation-induced pyroptosis, resulting in significant muscle loss, sarcopenia, and adverse skeletal muscle remodeling in diabetic muscle myopathy. Interventional treatment with BMP-7 attenuates hypercholesterolemia-induced inflammation-mediated sarcopenia and adverse muscle remodeling, suggesting BMP-7 could be a potential treatment option for diabetic muscle myopathy.

Using T1 mapping indices to evaluate muscle function and predict conservative treatment outcomes in diabetic patients with peripheral arterial disease.

To investigate interstitial muscle fibrosis via T1 mapping indices and its relationships with muscle function and conservative treatment outcomes. A total of 49 DM patients with PAD were prospectively recruited from 2016 to 2017. All PAD patients underwent pre-treatment MRI with conservative treatment via a rehabilitation program and antiplatelet therapy. The need to require percutaneous transluminal angioplasty intervention was recorded as intolerance to conservative treatment outcomes. We quantified calf interstitial muscle fibrosis using T1 mapping indices (native T1, post-contrast T1, and the extracellular volume fraction [ECV]). Muscle function was evaluated using a 6-min walking test (6MWT) and a 3-min stepping test (3MST). PAD patients were divided into two groups according to their tolerance or intolerance of the conservative treatment. Pearson's correlation, reproducibility, and multivariable Cox hazard analyses were performed with p < 0.05 indicating statistical significance. Among the T1 mapping indices in the posterior compartment of the calf in PAD patients, the native T1 value was significantly correlated with 6MWT (r = -0.422, p = 0.010) and 3MST (r = -0.427, p = 0.009). All T1 mapping indices showed excellent intra-observer and inter-observer correlations. ECV was an independent predictor of conservative treatment intolerance (average ECV, hazard ratio: 1.045, 95% confidence interval: 1.011-1.079, p = 0.009). T1 mapping measurements are reproducible with excellent intra-observer and inter-observer correlations. T1 mapping indices may be predictive of treatment and functional outcomes and carry promise in patient evaluation. Clinical Trials Identifier: NCT02850432 . • T1 mapping measurements of the calf muscles are reproducible with excellent intra-observer and inter-observer correlations (0.98 and 0.95 for anterior and posterior compartment muscle extracellular volume matrix [ECV] measurements, respectively). • ECV is shown to independently predict conservative treatment intolerance. • T1 mapping indices may be predictive of treatment and functional outcomes and carry promise in patient evaluation.

Next steps for the optimization of exon skipping therapy for Duchenne muscular dystrophy

Introduction It is established that the exon skipping approach can restore dystrophin in Duchenne muscular dystrophy (DMD) patients. However, dystrophin restoration levels are low, and the field is evolving to provide solutions for improved exon skipping. DMD is a neuromuscular disorder associated with chronic muscle tissue loss attributed to the lack of dystrophin, which causes muscle inflammation, fibrosis formation, and impaired regeneration. Currently, four antisense oligonucleotides (AONs) based on phosphorodiamidate morpholino oligomer (PMO) chemistry are approved by US Food and Drug Administration for exon skipping therapy of eligible DMD patients. Areas covered This review describes a preclinical and clinical experience with approved and newly developed AONs for DMD, outlines efforts that have been done to enhance AON efficiency, reviews challenges of clinical trials, and summarizes the current state of the exon skipping approach in the DMD field. Expert opinion The exon skipping approach for DMD is under development, and several chemical modifications with improved properties are under (pre)-clinical investigation. Despite existing advantages of these modifications, their safety and effectiveness have to be examined in clinical trials, which are planned or ongoing. Furthermore, we propose clinical settings using natural history controls to facilitate studying the functional effect of the therapy.

Temporal Changes in Key Signal Transduction Pathways Mediating Muscle Protein Synthesis with Adaptive and Maladaptive Right Ventricular Hypertrophy in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive disease characterized by occlusive remodeling of pulmonary arteries &lt; 500 micron and increased pulmonary vascular resistance. With the onset of PAH, the right ventricle (RV) of the heart adapts to the increased afterload pressure by undergoing adaptive hypertrophic remodeling to maintain adequate blood flow. However, for unknown reasons, maladaptive influences ensue, resulting in impaired RV function with progressive decompensation and right heart failure. Using a rodent model of PAH, key signaling pathways mediating cardiac muscle protein synthesis in the RV were evaluated during both the adaptive hypertrophy phase, with preserved right heart function, and the decompensated maladaptive phase, in which right heart failure (RHF) was present. Analysis of protein and gene expression changes in PAH animals identified three key signaling pathways involved in the shift toward maladaptive right heart failure: i) PI3K/Akt/mTOR; ii) GSK-3; iii) MAPK/ERK, as well as IGF-1 regulation. During adaptive hypertrophy, significant increments of phosphorylated proteins in the three signaling pathways were observed with increases in RV fibrosis and decreased capillarity found. In the maladaptive phase, mTORC1 and its downstream effector p-70S6K were significantly activated, contributing to the decreased LC3-I/II ratio, a marker of autophagy inhibition together with further significant RV muscle fibrosis and greater capillary rarefaction. We propose that autophagy inhibition in conjunction with other maladaptive processes reported in the decompensated RV muscle contributes to the genesis of overt RHF in PAH, and that a continuum of changes characterizes the adaptive and maladaptive phases in the RV muscle Keywords: Pulmonary Arterial Hypertension (P

Lack of concentration-dependent local toxicity of highly concentrated (5%) versus conventional 0.5% bupivacaine following musculoskeletal surgery in a rat model

PurposeVarious sustained-release formulations incorporate high bupivacaine concentrations but data on local toxicity is lacking. This study explores local toxic effects of highly concentrated (5%) bupivacaine compared to clinically used concentrations in vivo following skeletal surgery, to assess the safety of sustained-release formulations with high bupivacaine concentrations.MethodsSixteen rats underwent surgery, in which screws with catheters affixed were implanted in the spine or femur in a factorial experimental design, allowing single-shot or continuous 72 h local administration of 0.5%, 2.5% or 5.0% bupivacaine hydrochloride. During the 30-day follow-up, animal weight was recorded and blood samples were obtained. Implantation sites underwent histopathological scoring for muscle damage, inflammation, necrosis, periosteal reaction/thickening and osteoblast activity. Effects of bupivacaine concentration, administration mode and implantation site on local toxicity scores were analyzed.ResultsChi-squared tests for score frequencies revealed a concentration-dependent decrease in osteoblast count. Moreover, spinal screw implantation led to significantly more muscle fibrosis but less bone damage than femoral screw implantation, reflecting the more invasive muscle dissection and shorter drilling times related to the spinal procedure. No differences between bupivacaine administration modes regarding histological scoring or body weight changes were observed. Weight increased, while CK levels and leukocyte counts decreased significantly during follow-up, reflecting postoperative recovery. No significant differences in weight, leukocyte count and CK were found between interventional groups.ConclusionThis pilot study found limited concentration-dependent local tissue effects of bupivacaine solutions concentrated up to 5.0% following musculoskeletal surgery in the rat study population.

THE ULTRASONOGRAPHY EXAMINATION OF SKELETAL MUSCLES IN TRAUMATIC ISCHEMIA (EXPERIMENTAL STUDY).

The aim: To establish indicators and significance of sonography in the evaluation of muscle necrosis in ischemia of the limb acording to quantitative ultrasonographic indicators and density of collagen by histological method. Materials and methods: In experiments, rabbits modeled with 6-hour limb ischemia by applying an elastic tourniquet. On days 5, 15, and 30, ultrasound and histological studies of the muscles and correlation analysis were performed between the muscles' entropy and the degree of their damage (atrophy, fibrosis and necrosis). Results: The relative amount of structurally altered tissue was estimated morphometrically and compared with entropy. A high correlation of muscle damage with vertical δ-entropy indicates that sonography is highly likely to detect areas of necrosis and, to a lesser extent, fibrosis in the development of ischemic limb contracture in the early stages. Conclusions: Vertical δ-entropy in sonography is a significant indicator of muscle damage after traumatic ischemia and has strong relationship with muscle fibrosis.

Updates in the Use of Vamorolone and Steroids in the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating, life-limiting neuromuscular disorder that has a global prevelance of 21.4 cases per 100,000 live male births. DMD is caused by mutations in the dystrophin gene, which is integral to muscle membrane stability. Mutations in the dystrophin gene lead to inflammation and muscle fibrosis. Current standard-of-care treatments include the use of chronic (or classic) corticosteroids, which can cause significant side effects. Vamorolone is a first-in-class dissociative steroid that acts as a partial agonist to glucocorticoid receptors but as an antagonist to mineralocorticoid receptors, leading to a more favourable side effect profile. Clinical data has shown that vamorolone has similar anti-inflammatory benefits to classic corticosteroids but an improved side effect profile.

Osteopontin May Improve Postinjury Muscle Repair Via Matrix Metalloproteinases And tgf-β Activation in Regular Exercise.

Skeletal muscle injuries are commonly observed during sports and trauma. Regular exercise promotes muscle repair; however, the underlying mechanisms require further investigation. In addition to exercise, osteopontin (OPN) contributes to skeletal muscle regeneration and fibrosis following injury. However, whether and how OPN affects matrix proteins to promote post-injury muscle repair remains uncertain. We recruited regular exercise (RE) and sedentary control (SC) groups to determine plasma OPN levels. Additionally, we developed a murine model of muscle contusion injury and compared the extent of damage, inflammatory state, and regeneration-related proteins in OPN knockout (OPN KO) and wild-type (WT) mice. Our results show that regular exercise induced the increase of OPN, matrix metalloproteinases (MMPs), and transforming growth factor-β (TGF-β) expression in plasma. Injured muscle fibers were repaired more slowly in OPN-KO mice than in WT mice. The expression levels of genes and proteins related to muscle regeneration were lower in OPN-KO mice after injury. OPN also promotes fibroblast proliferation, differentiation, and migration. Additionally, OPN upregulates MMP expression by activating TGF-β, which promotes muscle repair. OPN can improve post-injury muscle repair by activating MMPs and TGF-β pathways. It is upregulated by regular exercise. Our study provides a potential target for the treatment of muscle injuries and explains why regular physical exercise is beneficial for muscle repair.

COVID-19 Mimics Pulmonary Dysfunction in Muscular Dystrophy as a Post-Acute Syndrome in Patients

Although progressive wasting and weakness of respiratory muscles are the prominent hallmarks of Duchenne muscular dystrophy (DMD) and long-COVID (also referred as the post-acute sequelae of COVID-19 syndrome); however, the underlying mechanism(s) leading to respiratory failure in both conditions remain unclear. We put together the latest relevant literature to further understand the plausible mechanism(s) behind diaphragm malfunctioning in COVID-19 and DMD conditions. Previously, we have shown the role of matrix metalloproteinase-9 (MMP9) in skeletal muscle fibrosis via a substantial increase in the levels of tumor necrosis factor-α (TNF-α) employing a DMD mouse model that was crossed-bred with MMP9-knockout (MMP9-KO or MMP9-/-) strain. Interestingly, recent observations from clinical studies show a robust increase in neopterin (NPT) levels during COVID-19 which is often observed in patients having DMD. What seems to be common in both (DMD and COVID-19) is the involvement of neopterin (NPT). We know that NPT is generated by activated white blood cells (WBCs) especially the M1 macrophages in response to inducible nitric oxide synthase (iNOS), tetrahydrobiopterin (BH4), and tetrahydrofolate (FH4) pathways, i.e., folate one-carbon metabolism (FOCM) in conjunction with epigenetics underpinning as an immune surveillance protection. Studies from our laboratory, and others researching DMD and the genetically engineered humanized (hACE2) mice that were administered with the spike protein (SP) of SARS-CoV-2 revealed an increase in the levels of NPT, TNF-α, HDAC, IL-1β, CD147, and MMP9 in the lung tissue of the animals that were subsequently accompanied by fibrosis of the diaphragm depicting a decreased oscillation phenotype. Therefore, it is of interest to understand how regulatory processes such as epigenetics involvement affect DNMT, HDAC, MTHFS, and iNOS that help generate NPT in the long-COVID patients.

Changes in pectoralis major stiffness and thickness following radiotherapy for breast cancer: A 12-month follow-up case series

Radiotherapy (RT) is a common and often essential treatment for breast cancer, but has been associated with pectoralis major (PM) muscle fibrosis and atrophy. In an initial prospective evaluation, we assessed muscle stiffness and muscle thickness of the sternocostal and clavicular regions of the PM with ultrasound shear wave elastography and B-mode imaging. Changes in PM muscle stiffness and thickness following RT can be detected within the first twelve months of RT completion. These parameters may potentially be useful for screening of patients who would benefit from post-RT physical therapy. Further studies with larger sample sizes that include patients who receive nodal radiation are necessary to confirm these findings.

Highland Barley Tea Polyphenols Extract Alleviates Skeletal Muscle Fibrosis in Mice by Reducing Oxidative Stress, Inflammation, and Cell Senescence.

The tea of roasted Highland barley is a cereal-based drink rich in polyphenols. A model of skeletal muscle senescence and fibrosis was constructed using d-galactose-induced C2C12 myotubes, and Highland barley tea Polyphenols (HBP) were extracted for the intervention. We found that HBP effectively alleviated oxidative stress, inflammation, and fibrosis induced by d-galactose-induced skeletal muscle senescence. Also, HBP treatment significantly down-regulated pro-fibrotic genes, inflammation, and oxidative stress levels in a contusion model of senescent mice. Reduced levels of SIRT3 protein was found to be an essential factor in skeletal muscle senescence and fibrosis in both cellular and animal models, while HBP treatment significantly increased SIRT3 protein levels and viability in skeletal muscle. The ability of HBP to mitigate skeletal muscle fibrosis and oxidative stress was significantly reduced after SIRT3 silencing. Together, these results suggest that HBP intervention can significantly alleviate aging-induced oxidative stress, inflammation, and skeletal muscle fibrosis, with the activation of SIRT3 as the underlying mechanism of action.

The Role of Matrix Metalloproteinase-13 (MMP13) in TGFβ/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

Muscle fibrosis and fatty infiltration (FI) are common complications seen in various muscle disease states. Recent studies indicate that muscle residential fibro/adipogenic progenitors (FAPs) are the major cellular source for muscle fibrosis and FI. We previously showed that MMP13 knockout (KO) mice have significantly increased FI, suggesting an important role of MMP13 in muscle FI. However, how MMP13 affects the differentiation of FAPs remains unknown. In order to assess the role of MMP-13 on FAP differentiation, we isolated FAPs from wildtype C57BL/6 and MMP13 knock out mice with FACS using CD31-, CD45-, Integrin α7- and Sca-1+ markers. FAPs were cultured in 24 well plate after FACS.in standard media till 80% confluent and then switched to adipogenic medium. In order to study the role of TGFβ and BMP in their differentiation, FAPs from both wildtype and MMP13 KO mice were treated with TGFβ1 (5 ng/ml). For MMP13 inhibitor treatment, FAPs from wildtype mice were incubated in adipogenic medium containing 10 µM MMP13 inhibitor (or vehicle) for 2 weeks. Immunofluorescence and gene expression analysis were used to assess FAP adipogenic and fibrogenic differentiation. FAPs were stained with Perilipin A (FITC, adipogenesis marker) and αSMA (Red, fibroblast marker), and DAPI. Real time PCR was performed for gene expression evaluation. A two-tailed Anova was used for statistical comparisons between groups, with p ≤ 0.05. Data are presented as mean ± standard deviation. In this study, we isolated FAPs from wildtype C57BL/6 and MMP13 KO mice and evaluated their adipogenic and fibrogenic differentiation in vitro. MMP13 KO FAPs demonstrated enhanced adipogenesis but reduced fibrogenesis compared to wildtype FAPs. Treating wildtype FAPs with an MMP13 inhibitor simulated phenotypes seen in MMP13 KO FAPs. In order to assess the role of MMP13 on TGFβ/BMP signaling in regulating FAP differentiation, we treated wildtype and MMP13 KO FAPs with TGFβ1, BMP7, TGFβ inhibitor, and BMP inhibitor. TGFβ1 treatment significantly enhanced fibrogenesis, but inhibited adipogenesis of wildtype FAPs. However, treatment with BMP7 showed the opposite effect. Interestingly, the effect of TGFβ1/BMP7 was voided in MMP13 KO FAPs. Treating wildtype FAPs with MMP13 inhibitor also abolished the effect of TGFβ1/BMP7 in FAP differentiation. Results from this study showed that TGFβ1 inhibits FAP adipogenesis but stimulates FAP fibrogenesis. BMP7 was shown to promote FAP adipogenesis but reduce its fibrogenesis. The role of the TGFβ/BMP signaling pathway regulating FAP differentiation was found to be MMP13 dependent. This study suggests that MMP13 is a critical downstream effector in TGFβ/BMP pathway which may serve as a new therapeutic target for muscle fibrosis and FI.

Microbiota dysbiosis influences immune system and muscle pathophysiology of dystrophin‐deficient mice

Duchenne muscular dystrophy (DMD) is a progressive severe muscle‐wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ‐free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.